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Pancreatic cancer (PC) is a lethal malignancy characterised by chemoresistance and metastatic spread. This is largely due to the presence of dense stromal fibrosis which distorts tumour vasculature. This limits nutrient, oxygen and chemotherapy perfusion into pancreatic tumours and promotes oxidative stress. The craftsmen of fibrosis are cancer-associated pancreatic stellate cells (CA-PSCs) who drive disease progression, chemoresistance and metastasis. To survive in the tumour microenvironment, CA-PSCs and PC cells undergo significant metabolic changes. A key alteration is increased expression of nutrient transporters to promote nutrient uptake. Solute carrier transporters have gained significant attention as a novel class of therapeutic targets, particularly solute carrier transporter 7A11 (SLC7A11) and solute carrier transporter 7A5 (SLC7A5), which are upregulated in PC cells and CA-PSCs. This study aimed: (1) to assess SLC7A11 inhibition in cancer-associated pancreatic stellate cells in vitro and in a clinically relevant orthotopic mouse model of PC; and (2) to investigate the function of SLC7A5 and SLC3A2 in cancer-associated pancreatic stellate cells in vitro. This study demonstrated that inhibiting SLC7A11 in CA-PSCs reduced proliferation. This limited cystine uptake, glutathione synthesis and anti-oxidant capacity of CA-PSCs and sensitised to oxidative stress. In a co-culture setting, inhibiting SLC7A11 in both PC cells and CA-PSCs significantly reduced colony number. We evaluated the therapeutic efficacy of two different SLC7A11 inhibitors, sulfasalazine and erastin. Sulfasalazine proved ineffective at reducing orthotopic pancreatic tumour growth, indicating it cannot be repurposed as PC therapy. Excitingly, erastin showed significant anti-proliferative effects in both PC cells and CA-PSCs. In CA-PSCs, the anti-proliferative effects were mediated by the induction of both ferroptosis and apoptosis. Lastly, we observed inhibition of SLC7A5 in CA-PSCs reduced p

Pancreatic cancer (PC) is a lethal disease due to chemoresistance and metastatic spread. We have shown that the microtubule protein βIII-tubulin is upregulated in PC cells, and that inhibition of βIII-tubulin (siRNA or shRNA) increased chemosensitivity in vitro and reduced tumour growth/metastases in vivo (McCarroll et al., Oncotarget, 6, pg.2235-49, 2015). Clinical translation of this work is limited by a lack of pharmacological inhibitors for βIII-tubulin. siRNA-based therapeutics are a potential solution, but require a delivery vehicle (e.g. nanoparticles). Novel star nanoparticles were designed to deliver siRNA to PC cells.The aims of this study were: 1) to design and optimise novel star-shaped nanoparticles that deliver functional siRNA to PC cells in vitro; 2) to examine the biodistribution and gene silencing activity of star-siRNA complexes in vivo using preclinical PC mouse models; and 3) to determine the potential of star-βIII-tubulin siRNA complexes to reduce tumour growth in preclinical PC mouse models. Results of this study demonstrated that star nanoparticles were able to complex and deliver βIII-tubulin siRNA to PC cells and potently silence βIII-tubulin expression. We modified our generation 1 nanoparticles by addition of PEGylation (Star-PEG) which enabled high gene silencing efficiency in PC cells in the presence of serum (an essential property for systemic administration in patients). All the star nanoparticles were non-toxic to PC cells and normal human pancreatic ductal cells. However, star-PEG nanoparticles displayed a superior toxicity profile compared to the non-PEGylated star nanoparticles. Star-PEG 4 (with 12.5% covalently attached PEG and longer cationic side-arms) was non-toxic in vivo and delivered siRNA to pancreatic tumours after systemic administration. Star-PEG 4-βIII-tubulin siRNA potently silenced βIII-tubulin in PC tumours and pilot studies indicated reduced tumour growth in both a subcutaneo

Inflammatory bowel diseases are chronic disorders of the gastrointestinal tract, consisting of ulcerative colitis and Crohn’s disease. These conditions are characterised by relapsing and remitting intestinal inflammation and they can lead to permanent, irreversible impairment of intestinal structure and function. There are strong human epidemiological data linking early appendicitis and subsequent removal of the inflamed appendix with a reduced risk of developing inflammatory bowel diseases later in life. We recapitulated this observation in a novel mouse model of appendicitis and trinitrobenzene sulfonic acid (TNBS)-induced colitis. Appendicitis-induced protection against colitis was found to be associated with preferential expansion of appendiceal regulatory T cells (Treg cells) and their migration to the colon and was dependent on the production of interleukin (IL)-10. The phenotypic characteristics of isolated colonic lamina propria (cLP) cells post appendicitis induction and the localisation of Treg cells in the colons of young appendicitis mice were examined. The mechanism regulating the preferential expansion of Treg cells was explored, by testing the hypothesis that retinoic acid (RA) is a key modulator of Treg cell expansion following appendicitis induction. First, immunohistochemical examination showed that the colonic lymphoid follicles (cLF) of appendicitis mice contained a large number of Foxp3+ T cells compared to their sparse distribution, which did not change after appendicitis induction, in the cLP. This suggests the cLFs are the potential site where Treg cells mediate their protective functions. Flow cytometry demonstrated that there was a significant increase in the proportion of CD4+Foxp3+CD25high Treg cells in the cLP of young mice and this change was restricted to mice in the appendicitis group (12.70% v.s 7.68%, p<0.04). Furthermore these Treg cells produce IL-10 (88.20% v.s 89.20%) and also expressed the chemokine receptors CCR6 (84.40% v.s 9

Pancreatic cancer is a leading cause of cancer-related deaths in Western societies. This poor prognosis is due to chemotherapeutic drug resistance and metastatic spread. Evidence suggests that microtubule proteins namely, beta-tubulins are dysregulated in tumor cells and are involved in regulating chemosensitivity. However, the role of beta-tubulins in pancreatic cancer are unknown. We measured the expression of different beta-tubulin isotypes in pancreatic adenocarcinoma tissue and pancreatic cancer cells. Next, we used RNAi to silence beta III-tubulin expression in pancreatic cancer cells, and measured cell growth in the absence and presence of chemotherapeutic drugs. Finally, we assessed the role of beta III-tubulin in regulating tumor growth and metastases using an orthotopic pancreatic cancer mouse model. We found that beta III-tubulin is highly expressed in pancreatic adenocarcinoma tissue and pancreatic cancer cells. Further, we demonstrated that silencing beta III-tubulin expression reduced pancreatic cancer cell growth and tumorigenic potential in the absence and presence of chemotherapeutic drugs. Finally, we demonstrated that suppression of beta III-tubulin reduced tumor growth and metastases in vivo. Our novel data demonstrate that beta III-tubulin is a key player in promoting pancreatic cancer growth and survival, and silencing its expression may be a potential therapeutic strategy to increase the long-term survival of pancreatic cancer patients., National Health and Medical Research Council (NHMRC); Cancer Council New South Wales; Cure Cancer Australia Foundation Grant; Cancer Institute NSW Fellowship; NHMRC CDF Fellowship; NHMRC Senior Research Fellowship

It is now generally accepted that activated pancreatic stellate cells (PSCs) are the principal effector cells in pancreatic fibrosis, a characteristic histological feature of chronic pancreatitis. PSC activation is associated with loss of cytoplasmic vitamin A containing lipid droplets, expression of cytoskeletal protein alpha-smooth muscle actin (αSMA), increased proliferation, and synthesis and secretion of extracellular matrix (ECM) proteins (the latter, in turn, influence gene expression patterns of PSCs during the activation process). One of the leading causes of chronic pancreatitis is alcohol abuse and in vitro studies have established that alcohol and cytokines (known to be released during pancreatic necroinflammation) can individually activate PSCs. However, during alcohol-induced pancreatic injury, PSCs are likely to be exposed to a combination of ethanol and cytokines (rather than individual factors).Therefore, aims of this study were 1) to investigate the combined effects of ethanol and cytokines on PSCs; 2) to assess and compare gene expression profiles of PSCs cultured on matrices that mimick normal and diseased pancreas - MatrigelTM and collagen I, respectively; 3) to validate and study the influence of selected genes (e.g. transgelin) on PSC activation.Results of this study demonstrated that i) at low doses, ethanol (5mM), IL-1 (0.5pg/mL) or TNFα (10U/mL) alone have negligible effects on PSC activation. However, PSCs treated with a combination of ethanol and cytokines exhibit significantly increased activation; ii) in PSCs cultured on different matrices (MatrigelTM, collagen I and plastic) several genes are dysregulated (fold change>2, p<0.001 and q<0.25) as follows: a) PSCs on collagen I vs MatrigelTM 146 dysregulated genes (76↓, 70↑); b) PSCs on matrigel vs plastic 619 dysregulated genes (297↓, 322↑); and c) PSCs on collagen I vs plastic 432 dysregulated genes (178↓, 254↑); iii) selected genes (transgel

Pancreatic stellate cells (PSCs) produce the stromal reaction of pancreatic cancer (PC) and their interaction with cancer cells facilitates cancer progression. This study investigated the role of human PSCs (hPSCs) in the metastatic process and tumor angiogenesis using an in vivo (orthotopic model) and in vitro (cultured PSC and PC cells) approach. A gender mismatch study [injection of male hPSCs + female PC cells into the pancreas of female mice] was conducted to determine whether hPSCs accompany cancer cells to metastatic sites. Metastatic nodules were examined by fluorescent in situ hybridization for the presence of the y chromosome. Angiogenesis was assessed by i) immunostaining tumors for CD31, an endothelial cell marker; and ii) in vitro quantifying human microvascular endothelial cell (HMEC-1) tube formation upon exposure to conditioned media from hPSCs. Transendothelial migration was assessed by examining the movement of fluorescently labeled hPSCs through an endothelial cell monolayer. Human PSCs i) were found in multiple metastatic sites in each mouse injected with male hPSCs + female PC cells; ii) increased CD31 expression in primary tumors from mice injected with MiaPaCa-2 and hPSCs and stimulated tube formation by HMEC-1 in vitro; iii) exhibited transendothelial migration which was stimulated by cancer cells. Human PSCs accompany cancer cells to metastatic sites, stimulate angiogenesis and have the capacity to intravasate/extravasate to and from blood vessels.